Ngaphambili besixele ukuba amanqanaba e-serum ye-tryptophan metabolite indole-3-propionic acid (IPA) evela kwi-gut-derived asezantsi kwizigulane ezine-liver fibrosis. Kolu phononongo, siphande i-transcriptome kunye ne-DNA methylome kwi-epidermis etyebileyo ngokumalunga namanqanaba e-IPA eserum, kunye nendima ye-IPA ekukhuthazeni ukungasebenzi kwe-phenotypic kwee-hepatic stellate cells (HSCs) kwi-vitro.
Olu phononongo luquke izigulana ezili-116 ezityebileyo ezingenaso isifo seswekile sohlobo lwesibini (T2DM) (ubudala obungama-46.8 ± 9.3 iminyaka; i-BMI: 42.7 ± 5.0 kg/m²) ezenze utyando lwe-bariatric kwiZiko loPhando lwe-Kuopio Bariatric (KOBS). Amanqanaba e-IPA ajikelezileyo alinganiswe nge-liquid chromatography-mass spectrometry (LC-MS), uhlalutyo lwe-liver transcriptome lwenziwe nge-total RNA sequencing, kwaye uhlalutyo lwe-DNA methylation lwenziwa kusetyenziswa i-Infinium HumanMethylation450 BeadChip. Iiseli ze-hepatic stellate zabantu (LX-2) zisetyenziselwe uvavanyo lwe-in vitro.
Amanqanaba e-IPA e-serum anxulumene nokubonakaliswa kweejini ezibandakanyekayo kwiindlela ze-apoptotic, mitophagic, kunye ne-longitude kwisibindi. I-gene ye-AKT serine/threonine kinase 1 (AKT1) yayiyeyona jini ininzi neyilawulayo kwi-liver transcript kunye neeprofayili ze-DNA methylation. Unyango lwe-IPA lwabangela i-apoptosis, lwanciphisa ukuphefumla kwe-mitochondrial, lwaza lwatshintsha imo yeseli kunye ne-mitochondrial dynamics ngokuguqula ukubonakaliswa kweejini ezaziwa ngokulawula i-fibrosis, i-apoptosis, kunye nokusinda kweeseli ze-LX-2.
Xa zizonke, ezi datha zixhasa ukuba i-IPA ineziphumo zonyango ezinokubakho kwaye inokubangela i-apoptosis kwaye itshintshe i-phenotype ye-HSC iye kwimeko engasebenziyo, ngaloo ndlela yandisa amathuba okuthintela i-fibrosis yesibindi ngokuphazamisa ukusebenza kwe-HSC kunye ne-metabolism ye-mitochondrial.
Ukuxhaphaka kwesifo sokutyeba kakhulu kunye nesifo se-metabolic syndrome kuye kwanxulunyaniswa nokwanda kwesifo sesibindi esinamafutha esinxulumene ne-metabolically associated fatty liver disease (MASLD); esi sifo sichaphazela i-25% ukuya kwi-30% yoluntu ngokubanzi [1]. Isiphumo esiphambili se-MASLD etiology yi-liver fibrosis, inkqubo eguqukayo ebonakaliswa kukuqokelelwa okuqhubekayo kwe-fibrous extracellular matrix (ECM) [2]. Iiseli eziphambili ezibandakanyeka kwi-liver fibrosis zii-hepatic stellate cells (HSCs), ezibonisa ii-phenotypes ezine ezaziwayo: ezithuleyo, ezivuselelweyo, ezingasebenziyo, kunye nezisenenescent [3, 4]. Ii-HSC zingasetyenziswa kwaye zitshintshe ukusuka kwimo ethuleyo ziye kwiiseli ezifana ne-fibroblast ezikhulayo ezineemfuno zamandla aphezulu, kunye nokubonakaliswa okwandisiweyo kwe-α-smooth muscle actin (α-SMA) kunye ne-type I collagen (Col-I) [5, 6]. Ngexesha lokuguqulwa kwe-liver fibrosis, ii-HSC ezisebenzayo zisuswa nge-apoptosis okanye i-inactivation. Ezi nkqubo ziquka ukunciphisa iijini ze-fibrogenic kunye nokuguqulwa kweejini ze-prosurvival (ezifana neendlela ze-NF-κB kunye ne-PI3K/Akt signaling) [7, 8], kunye notshintsho kwi-mitochondrial dynamics kunye nomsebenzi [9].
Amanqanaba e-serum ye-tryptophan metabolite indole-3-propionic acid (IPA), eveliswa emathunjini, afunyenwe ehla kwizifo ze-metabolic zabantu kuquka i-MASLD [10-13]. I-IPA inxulunyaniswa nokutya i-fiber, yaziwa ngemiphumo yayo ye-antioxidant kunye ne-anti-inflammatory, kwaye inciphisa i-phenotype ye-steatohepatitis (NASH) ebangelwa kukutya kwi-vivo nakwi-in vitro [11-14]. Ubungqina obuthile buvela kuphando lwethu lwangaphambili, olubonise ukuba amanqanaba e-IPA eserum ayephantsi kwizigulane ezine-fibrosis yesibindi kunakwizigulane ezityebileyo ezingenayo i-fibrosis yesibindi kwi-Kuopio Bariatric Surgery Study (KOBS). Ngaphezu koko, sibonise ukuba unyango lwe-IPA lunokunciphisa ukubonakaliswa kwee-genes eziziimpawu zakudala zokunamathela kweseli, ukufuduka kweseli kunye nokusebenza kwe-hematopoietic stem cell kwimodeli ye-human hepatic stellate cell (LX-2) kwaye yi-metabolite enokwenzeka ye-hepatoprotective [15]. Nangona kunjalo, akukacaci ukuba i-IPA ibangela njani ukuhlehla kwe-fibrosis yesibindi ngokusebenzisa i-HSC apoptosis kunye ne-mitochondrial bioenergetics.
Apha, sibonisa ukuba i-IPA yeserum inxulunyaniswa nokubonakaliswa kwezakhi zofuzo ezityebiswe kwi-apoptosis, i-mitophagy, kunye neendlela zokuphila ixesha elide kwisibindi sabantu abatyebe kakhulu kodwa abangengohlobo lwesibini lwesifo seswekile (KOBS). Ngaphezu koko, sifumanise ukuba i-IPA inokubangela ukususwa kunye nokuwohloka kwee-activated hematopoietic stem cells (HSCs) ngendlela yokungasebenzi. Ezi ziphumo zibonisa indima entsha ye-IPA, nto leyo eyenza ukuba ibe yindawo ekujoliswe kuyo yonyango ukukhuthaza ukwehla kwe-fibrosis yesibindi.
Uphononongo lwangaphambili kwiqela le-KOBS lubonise ukuba izigulana ezine-fibrosis yesibindi zazinamanqanaba aphantsi e-IPA xa kuthelekiswa nezigulana ezingenayo i-fibrosis yesibindi [15]. Ukuze sikhuphe isiphumo esinokubangela ukudideka sesifo seswekile sohlobo lwesibini, siqeshe izigulana ezili-116 ezityebileyo ezingenaso isifo seswekile sohlobo lwesibini (ubudala obuphakathi ± SD: 46.8 ± 9.3 iminyaka; BMI: 42.7 ± 5.0 kg/m2) (Itheyibhile 1) kwisifundo se-KOBS esiqhubekayo njengabantu abafundayo [16]. Bonke abathathi-nxaxheba banike imvume ebhaliweyo kwaye inkqubo yophando yavunywa yiKomiti yoBulungisa yeSibhedlele saseNorth Savo County ngokuhambelana neSibhengezo saseHelsinki (54/2005, 104/2008 kunye no-27/2010).
Iisampulu ze-biopsy yesibindi zifunyenwe ngexesha lotyando lwe-bariatric kwaye zahlolwa ngokwe-histological ziingcali ze-pathologists ezinamava ngokweekhrayitheriya ezichazwe ngaphambili [17, 18]. Iikhrayitheriya zovavanyo zishwankathelwe kwiTheyibhile eyoNgezelelweyo S1 kwaye zichazwe ngaphambili [19].
Iisampulu ze-serum yokuzila ukutya zihlalutywe nge-untargeted liquid chromatography-mass spectrometry (LC-MS) yohlalutyo lwe-metabolomics (n = 116). Iisampulu zihlalutywe kusetyenziswa inkqubo ye-UHPLC-qTOF-MS (1290 LC, 6540 qTOF-MS, Agilent Technologies, Waldbronn, Karlsruhe, Germany) njengoko kuchaziwe ngaphambili19. Ukuchongwa kwe-isopropyl alcohol (IPA) kwakusekelwe kwixesha lokugcina kunye nokuthelekiswa kwe-MS/MS spectrum nemigangatho ecocekileyo. Ubunzulu besignali ye-IPA (indawo ephakame kakhulu) buqwalaselwe kuzo zonke ezinye iinkcazo [20].
Ulandelelwano lwe-RNA yesibindi siphela lwenziwe kusetyenziswa i-Illumina HiSeq 2500 kwaye idatha yalungiswa kwangaphambili njengoko kuchaziwe ngaphambili [19, 21, 22]. Senze uhlalutyo lokuchazwa komahluko olujoliswe ekuchazeni ii-transcripts ezichaphazela umsebenzi we-mitochondrial/biogenesis sisebenzisa ii-genes ze-1957 ezikhethwe kwisiseko sedatha seMitoMiner 4.0 [23]. Uhlalutyo lwe-methylation ye-DNA yesibindi lwenziwe kusetyenziswa i-Infinium HumanMethylation450 BeadChip (Illumina, San Diego, CA, USA) kusetyenziswa indlela efanayo naleyo ichaziwe ngaphambili [24, 25].
Iiseli ze-hepatic stellate zabantu (LX-2) zibonelelwe ngobubele nguNjingalwazi Stefano Romeo kwaye zakhuliswa zaza zagcinwa kwi-DMEM/F12 medium (Biowest, L0093-500, 1% Pen/Strep; Lonza, DE17-602E, 2% FBS; Gibco, 10270-106). Ukukhetha idosi yokusebenza ye-IPA, iiseli ze-LX-2 zanyangwa ngamaqondo ahlukeneyo e-IPA (10 μM, 100 μM kunye ne-1 mM; Sigma, 220027) kwi-DMEM/F12 medium kangangeeyure ezingama-24. Ngaphezu koko, ukuphanda amandla e-IPA okuvala ii-HSC, iiseli ze-LX-2 zanyangwa kunye ne-5 ng/ml TGF-β1 (iinkqubo ze-R&D, 240-B-002/CF) kunye ne-1 mM IPA kwi-serum-free medium kangangeeyure ezingama-24. Kwizixhobo zokulawula izithuthi ezifanelekileyo, i-4 nM HCL equlethe i-0.1% BSA yasetyenziswa kunyango lwe-TGF-β1 kwaye i-0.05% DMSO yasetyenziswa kunyango lwe-IPA, kwaye zombini zasetyenziswa kunye kunyango oludibeneyo.
I-Apoptosis ihlolwe kusetyenziswa i-FITC Annexin V Apoptosis Detection Kit ene-7-AAD (Biolegend, San Diego, CA, USA, Cat# 640922) ngokwemiyalelo yomenzi. Ngokufutshane, i-LX-2 (1 × 105 cells/well) yakhuliswa ubusuku bonke kwiiplate ze-12-well emva koko yanyangwa ngeedosi ezininzi ze-IPA okanye i-IPA kunye ne-TGF-β1. Ngosuku olulandelayo, iiseli ezidadayo nezinamathelayo zaqokelelwa, zatshintshwa, zahlanjwa nge-PBS, zaphinda zaxhonywa kwi-Annexin V binding buffer, zaza zafakwa kwi-FITC-Annexin V kunye ne-7-AAD imizuzu eli-15.
I-Mitochondria kwiiseli eziphilayo yafakwa umbala ngenxa yomsebenzi we-oxidative kusetyenziswa i-Mitotracker™ Red CMXRos (MTR) (Thermo Fisher Scientific, Carlsbad, CA). Kwiimvavanyo ze-MTR, iiseli ze-LX-2 zafakwa kwi-densities elinganayo ne-IPA kunye ne-TGF-β1. Emva kweeyure ezingama-24, iiseli eziphilayo zafakwa kwi-trypsin, zahlanjwa nge-PBS, zaza zafakwa kwi-incubator nge-100 μM MTR kwindawo engena-serum kwi-37 °C imizuzu engama-20 njengoko kuchaziwe ngaphambili [26]. Kuhlalutyo lwe-morphology yeseli ephilayo, ubungakanani beseli kunye nobunzima be-cytoplasmic buhlalutywe kusetyenziswa iiparameter ze-forward scatter (FSC) kunye ne-side scatter (SSC), ngokulandelanayo.
Yonke idatha (iziganeko ezingama-30,000) ifunyenwe kusetyenziswa iNovoCyte Quanteon (Agilent) kwaye yahlalutywa kusetyenziswa iNovoExpress® 1.4.1 okanye isoftware yeFlowJo V.10.
Izinga lokusetyenziswa kweoksijini (OCR) kunye nezinga le-acidification yangaphandle (ECAR) zilinganiswe ngexesha langempela kusetyenziswa i-Seahorse Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA) exhotyiswe nge-Seahorse XF Cell Mito Stress ngokwemiyalelo yomenzi. Ngokufutshane, ii-2 × 104 LX-2 cells/well zatyalwa kwiiplate ze-XF96 cell culture. Emva kokufunxwa ebusuku, ii-cells zanyangwa nge-isopropanol (IPA) kunye ne-TGF-β1 (Iindlela ezongezelelweyo 1). Uhlalutyo lwedatha lwenziwe kusetyenziswa isoftware ye-Seahorse XF Wave, equka i-Seahorse XF Cell Energy Phenotype Test Report Generator. Ukusuka koku, kwabalwa i-Bioenergetic Health Index (BHI) [27].
I-RNA iyonke iguqulelwe kwi-cDNA. Ukuze ufumane iindlela ezithile, jonga ireferensi [15]. Amanqanaba e-mRNA e-ribosomal acidic protein P0 (RPLP0) kunye ne-cyclophilin A1 (PPIA) yabantu asetyenziswe njengolawulo lwezakhi zofuzo. Inkqubo ye-QuantStudio 6 pro Real-Time PCR (Thermo Fisher, Landsmeer, The Netherlands) isetyenzisiwe kunye ne-TaqMan™ Fast Advanced Master Mix Kit (Applied Biosystems) okanye i-Sensifast SYBR Lo-ROX Kit (Bioline, BIO 94050), kunye ne-relative gene expression fold ibalwe kusetyenziswa iiparamitha ze-Ct value cycling parameters (ΔΔCt) kunye nendlela ye-∆∆Ct. Iinkcukacha zee-primers zibonelelwe kwiTheyibhile eziNcedisayo S2 kunye ne-S3.
I-Nuclear DNA (ncDNA) kunye ne-mitochondrial DNA (mtDNA) zikhutshwe kusetyenziswa i-DNeasy blood and tissue kit (Qiagen) njengoko kuchaziwe ngaphambili [28]. Isixa esithelekisekayo se-mtDNA sibalwe ngokubala umlinganiselo wendawo nganye ekujoliswe kuyo ye-mtDNA kwi-geometric mean yemimandla emithathu ye-nuclear DNA (mtDNA/ncDNA), njengoko kuchaziwe kwiindlela ezongezelelweyo 2. Iinkcukacha zee-primers ze-mtDNA kunye ne-ncDNA zibonelelwe kwiTheyibhile eyongezelelweyo S4.
Iiseli eziphilayo zafakwa i-Mitotracker™ Red CMXRos (MTR) (Thermo Fisher Scientific, Carlsbad, CA) ukuze zibone iinethiwekhi ze-mitochondrial eziphakathi kweeseli kunye nezangaphakathi kweeseli. Iiseli ze-LX-2 (1 × 104 iiseli/umngxuma) zakhuliswa kwiislayidi zeglasi kwiiplate zenkcubeko ezihambelanayo ezinezantsi kweglasi (Ibidi GmbH, Martinsried, eJamani). Emva kweeyure ezingama-24, iiseli ze-LX-2 eziphilayo zafakwa i-100 μM MTR kangangemizuzu engama-20 kwi-37 °C kwaye ii-nuclei zeeseli zafakwa i-DAPI (1 μg/ml, Sigma-Aldrich) njengoko kuchaziwe ngaphambili [29]. Iinethiwekhi ze-Mitochondrial zafakwa i-microscope eguqulweyo ye-Zeiss Axio Observer (Carl Zeiss Microimaging GmbH, Jena, eJamani) exhotyiswe nge-Zeiss LSM 800 confocal module kwi-37 °C kwindawo enomswakama ene-5% CO2 kusetyenziswa injongo ye-63×NA 1.3. Sifumene imifanekiso elishumi yochungechunge lwe-Z kuhlobo ngalunye lwesampulu. Uthotho ngalunye lwe-Z luqulathe amacandelo angama-30, ngalinye linobukhulu obuyi-9.86 μm. Kwisampulu nganye, imifanekiso yeendawo ezilishumi ezahlukeneyo zokujonga ifunyenwe kusetyenziswa isoftware ye-ZEN 2009 (Carl Zeiss Microimaging GmbH, Jena, Germany), kwaye uhlalutyo lwe-mitochondrial morphology lwenziwe kusetyenziswa isoftware ye-ImageJ (v1.54d) [30, 31] ngokweeparameters ezichazwe kwiSupplementary Methods 3.
Iiseli zilungisiwe nge-2% glutaraldehyde kwi-0.1 M phosphate buffer, kulandele ukufakelwa ngesisombululo se-osmium tetroxide esingu-1% (Sigma Aldrich, MO, USA), zancitshiswa kancinci kancinci nge-acetone (Merck, Darmstadt, Germany), zaza ekugqibeleni zafakwa kwi-epoxy resin. Amacandelo amancinci kakhulu alungiswa kwaye adaywa nge-1% uranyl acetate (Merck, Darmstadt, Germany) kunye ne-1% lead citrate (Merck, Darmstadt, Germany). Imifanekiso ye-Ultrastructural ifunyenwe kusetyenziswa i-JEM 2100F EXII transmission electron microscope (JEOL Ltd, Tokyo, Japan) kwi-voltage ekhawulezayo ye-80 kV.
Imo yeeseli ze-LX-2 ezinyangwe nge-IPA kangangeeyure ezingama-24 ihlalutywe nge-phase-contrast microscopy kwi-50x magnification kusetyenziswa i-Zeiss inverted light microscope (Zeiss Axio Vert.A1 kunye ne-AxioCam MRm, eJena, eJamani).
Idatha yeklinikhi ichazwe njenge-mean ± standard deviation okanye i-median (interquartile range: IQR). Uhlalutyo lwendlela enye lokwahluka (continuous variables) okanye uvavanyo lwe-χ² (categorical variables) lusetyenzisiwe ukuthelekisa umahluko phakathi kwamaqela amathathu ophando. I-false positive rate (FDR) isetyenzisiwe ukulungisa uvavanyo oluninzi, kwaye ii-genes ezine-FDR <0.05 zithathwe njengezibalulekileyo ngokwezibalo. Uhlalutyo lwe-Spearman correlation lusetyenzisiwe ukudibanisa i-CpG DNA methylation kunye nobunzulu besignali ye-IPA, kunye ne-nominal p values ​​​​(p <0.05) ezixeliweyo.
Uhlalutyo lwendlela lwenziwe kusetyenziswa isixhobo sohlalutyo lweseti yezakhi zofuzo esisekelwe kwiwebhu (iWebGestalt) kwiitranscript ezingama-268 (i-nominal p < 0.01), iitranscript ezidityaniswe ne-mitochondria ezili-119 (i-nominal p < 0.05), kunye ne-4350 CpGs kwiitranscript zesibindi ezingama-3093 ezazidibene namanqanaba e-IPA eserum ajikelezayo. Isixhobo seVenny DB (version 2.1.0) esifumaneka simahla sasetyenziswa ukufumana izakhi zofuzo ezihambelanayo, kwaye iStringDB (version 11.5) yasetyenziswa ukubonisa unxibelelwano lweproteni neproteni.
Kwilinge le-LX-2, iisampulu zavavanywa ukuze zibonakale ziqhelekile kusetyenziswa uvavanyo lwe-D'Agostino-Pearson. Idatha ifunyenwe kwiikopi ezintathu zebhayoloji kwaye yafakwa kwi-ANOVA yendlela enye ngovavanyo lwe-Bonferroni post hoc. Ixabiso le-p elingaphantsi kwe-0.05 lithathwe njengelibalulekileyo ngokwezibalo. Idatha iboniswa njenge-mean ± SD, kwaye inani leemvavanyo liboniswe kumfanekiso ngamnye. Zonke iihlalutyo kunye neegrafu zenziwe kusetyenziswa isoftware yezibalo yeGraphPad Prism 8 yeWindows (GraphPad Software Inc., inguqulelo 8.4.3, eSan Diego, e-USA).
Okokuqala, siphande unxulumano lwamanqanaba e-IPA eserum kunye nesibindi, umzimba wonke, kunye ne-mitochondrial transcripts. Kwiprofayili iyonke ye-transcript, i-gene enamandla enxulunyaniswa namanqanaba e-IPA eserum yayiyi-MAPKAPK3 (FDR = 0.0077; i-protein kinase-activated protein kinase-activated protein kinase 3); kwiprofayili ye-transcript enxulumene ne-mitochondria, i-gene enamandla enxulunyaniswayo yayiyi-AKT1 (FDR = 0.7621; AKT serine/threonine kinase 1) (Ifayile eyongezelelweyo 1 kunye nefayile eyongezelelweyo 2).
Emva koko sihlalutye ii-transcript zehlabathi (n = 268; p < 0.01) kunye nee-transcript ezinxulumene ne-mitochondria (n = 119; p < 0.05), ekugqibeleni sichonga i-apoptosis njengeyona ndlela ibalulekileyo ye-canonical (p = 0.0089). Kwii-transcript ze-mitochondrial ezinxulumene namanqanaba e-IPA eserum, sigxile kwi-apoptosis (FDR = 0.00001), i-mitophagy (FDR = 0.00029), kunye neendlela ze-TNF signaling (FDR = 0.000006) (Umfanekiso 1A, Itheyibhile 2, kunye neMifanekiso eyongezelelweyo 1A-B).
Uhlalutyo oluhambelanayo lwe-translator yehlabathi, enxulumene ne-mitochondria, kunye ne-methylation ye-DNA kwisibindi somntu ngokunxulumene namanqanaba e-IPA eserum. I-A imele ii-translator zehlabathi ezingama-268, ii-translator ezinxulumene ne-mitochondria ezili-119, kunye nee-translator ze-DNA methylated ezidweliswe kwiindawo ezingama-3092 ze-CpG ezinxulumene namanqanaba e-IPA eserum (amaxabiso e-p < 0.01 kwii-translator zehlabathi kunye ne-DNA methylated, kunye namaxabiso e-p < 0.05 kwii-translator ze-mitochondrial). Ii-translator eziphambili ezihambelanayo ziboniswe embindini (AKT1 kunye ne-YKT6). B Imephu yokusebenzisana kwee-genes ezili-13 ezinenqaku eliphezulu lokusebenzisana (0.900) nezinye ii-genes yakhiwe kwii-genes ezingama-56 ezihambelanayo (ummandla omnyama) ezazidibene kakhulu namanqanaba e-IPA eserum kusetyenziswa isixhobo esikwi-intanethi i-StringDB. Iluhlaza: Ii-genes ezidweliswe kwi-Gene Ontology (GO) cell component: mitochondria (GO:0005739). I-AKT1 yiproteni enamanqaku aphezulu (0.900) okunxibelelana nezinye iiproteni ngokusekelwe kwidatha (esekelwe ekufumaneni umbhalo, iimvavanyo, iidathabheyisi, kunye nokubonakaliswa ngokubambisana). Ama-node enethiwekhi amele iiproteni, kwaye imiphetho imele unxibelelwano phakathi kweeproteni.
Ekubeni i-gut microbiota metabolites inokulawula ukwakheka kwe-epigenetic nge-DNA methylation [32], siphande ukuba amanqanaba e-IPA eserum adibene ne-methylation ye-DNA yesibindi. Sifumanise ukuba iindawo ezimbini eziphambili ze-methylation ezinxulumene namanqanaba e-IPA eserum zazikufutshane ne-proline-serine-rich region 3 (C19orf55) kunye ne-heat shock protein family B (small) member 6 (HSPB6) (Ifayile eyongezelelweyo 3). I-DNA methylation ye-4350 CpG (p < 0.01) yayinxulumene namanqanaba e-IPA eserum kwaye yatyetyiswa kwiindlela zokulawula ixesha elide (p = 0.006) (Umfanekiso 1A, Itheyibhile 2, kunye noMfanekiso ongezelelweyo 1C).
Ukuze siqonde iindlela zebhayoloji ezisisiseko sobudlelwane phakathi kwamanqanaba e-IPA eserum, ii-transcript zehlabathi, ii-transcript ezinxulumene ne-mitochondria, kunye ne-methylation ye-DNA kwisibindi somntu, senze uhlalutyo oluhambelanayo lwee-genes ezichongiweyo kuhlalutyo lwendlela yangaphambili (Umfanekiso 1A). Iziphumo zohlalutyo lokuphucula indlela yee-genes ezingama-56 ezigqubanayo (ngaphakathi komgca omnyama kuMfanekiso 1A) zibonise ukuba indlela ye-apoptosis (p = 0.00029) iqaqambise ii-genes ezimbini ezifanayo kwii-analysis ezintathu: i-AKT1 kunye ne-YKT6 (YKT6 v-SNARE homolog), njengoko kubonisiwe kumzobo weVenn (Umfanekiso ongezelelweyo 2 kunye noMfanekiso 1A). Okunomdla kukuba, sifumanise ukuba i-AKT1 (cg19831386) kunye ne-YKT6 (cg24161647) zinxulumene kakuhle namanqanaba e-IPA eserum (Ifayile eyongezelelweyo 3). Ukuze sichonge ukusebenzisana okunokwenzeka kweeproteni phakathi kweemveliso ze-gene, sikhethe ii-genes ezili-13 ezine-common region score ephezulu (0.900) phakathi kwe-56 ii-genes ezigqubanayo njenge-input kwaye sakha imephu yokusebenzisana. Ngokwenqanaba lokuzithemba (ukuzithemba okungaphantsi), i-AKT1 gene enenqaku eliphezulu (0.900) yayiphezulu (Umfanekiso 1B).
Ngokusekelwe kuhlalutyo lwendlela, sifumanise ukuba i-apoptosis yeyona ndlela iphambili, ngoko ke siphande ukuba unyango lwe-IPA luya kuyichaphazela na i-apoptosis ye-HSCs kwi-vitro. Ngaphambili sibonise ukuba iidosi ezahlukeneyo ze-IPA (10 μM, 100 μM, kunye ne-1 mM) azinabungozi kwiiseli ze-LX-2 [15]. Olu phononongo lubonise ukuba unyango lwe-IPA kwi-10 μM kunye ne-100 μM lonyusa inani leeseli ezisebenzayo nezifileyo. Nangona kunjalo, xa kuthelekiswa neqela lolawulo, ukuphila kweseli kwehla kwi-1 mM IPA concentration, ngelixa izinga le-cell necrosis lihlala lingatshintshanga (Umfanekiso 2A, B). Okulandelayo, ukuze sifumane uxinaniso olufanelekileyo lokukhuthaza i-apoptosis kwiiseli ze-LX-2, sivavanye i-10 μM, 100 μM, kunye ne-1 mM IPA kangangeeyure ezingama-24 (Umfanekiso 2A-E kunye noMfanekiso ongezelelweyo 3A-B). Okunomdla kukuba, i-IPA 10 μM kunye ne-100 μM zehlise izinga le-apoptosis (%), nangona kunjalo, i-IPA 1 mM yonyuse izinga le-apoptosis kunye ne-apoptosis (%) xa kuthelekiswa nolawulo kwaye ke ngoko yakhethwa kwiimvavanyo ezongezelelweyo (Imifanekiso 2A–D).
I-IPA ibangela i-apoptosis yeeseli ze-LX-2. Indlela ye-Annexin V kunye ne-7-AAD yokudaya kabini isetyenzisiwe ukulinganisa izinga le-apoptotic kunye ne-cell morphology nge-flow cytometry. Iiseli ze-BA zafakwa kwi-10 μM, 100 μM kunye ne-1 mM IPA kangangeeyure ezingama-24 okanye nge-F–H TGF-β1 (5 ng/ml) kunye ne-1 mM IPA kwindawo engena-serum kangangeeyure ezingama-24. A: iiseli eziphilayo (Annexin V -/ 7AAD-); B: iiseli ze-necrotic (Annexin V -/ 7AAD+); C, F: kwangoko (Annexin V +/ 7AAD-); D, G: emva kwexesha (Annexin V+/7AAD+.); E, H: ipesenti yeeseli ze-apoptotic zakuqala nezemva kwexesha kwizinga le-apoptotic (%). Idatha ichazwa njenge-avareji ± SD, n = iimvavanyo ezi-3 ezizimeleyo. Uthelekiso lwezibalo lwenziwe kusetyenziswa i-ANOVA yendlela enye kunye novavanyo lwe-Bonferroni emva kwe-hoc. *p < 0.05; ****p < 0.0001
Njengoko besibonisile ngaphambili, i-5 ng/ml TGF-β1 inokubangela ukusebenza kwe-HSC ngokwandisa ukubonakaliswa kwe-classical marker genes [15]. Iiseli ze-LX-2 ziphathwe nge-5 ng/ml TGF-β1 kunye ne-1 mM IPA ngokudibeneyo (Umzobo 2E–H). Unyango lwe-TGF-β1 aluzange lutshintshe izinga le-apoptosis, nangona kunjalo, unyango lwe-IPA oludibeneyo lonyuse izinga le-apoptosis kunye ne-apoptosis (%) xa kuthelekiswa nonyango lwe-TGF-β1 (Umzobo 2E–H). Ezi ziphumo zibonisa ukuba i-1 mM IPA inokukhuthaza i-apoptosis kwiiseli ze-LX-2 ngokuzimeleyo ngaphandle kokungeniswa kwe-TGF-β1.
Siqhubele phambili ngophando lwethu ngempembelelo ye-IPA ekuphefumleni kwe-mitochondrial kwiiseli ze-LX-2. Iziphumo zibonise ukuba i-1 mM IPA yehlise iiparameter zezinga lokusetyenziswa kweoksijini (OCR): ukuphefumla okungekho mitochondrial, ukuphefumla okusisiseko kunye nokuphezulu, ukuvuza kweproton kunye nemveliso ye-ATP xa kuthelekiswa neqela lolawulo (Umfanekiso 3A, B), ngelixa i-bioenergetic health index (BHI) ingatshintshanga.
I-IPA inciphisa ukuphefumla kwe-mitochondrial kwiiseli ze-LX-2. I-mitochondrial respiration curve (OCR) iboniswa njengeeparameter zokuphefumla kwe-mitochondrial (ukuphefumula okungeyiyo i-mitochondrial, ukuphefumla kwe-basal, ukuphefumla okuphezulu, ukuvuza kweproton, ukuveliswa kwe-ATP, i-SRC kunye ne-BHI). Iiseli A kunye ne-B zifakwe i-10 μM, i-100 μM kunye ne-1 mM IPA kangangeeyure ezingama-24, ngokulandelelana. Iiseli C kunye ne-D zifakwe i-TGF-β1 (5 ng/ml) kunye ne-1 mM IPA kwi-serum-free medium kangangeeyure ezingama-24, ngokulandelelana. Zonke izilinganiso zilungisiwe ngokomgangatho we-DNA kusetyenziswa i-CyQuant kit. I-BHI: i-bioenergetic health index; i-SRC: umthamo wogcino lokuphefumla; i-OCR: izinga lokusetyenziswa kwe-oxygen. Idatha iboniswa njenge-mean ± standard deviation (SD), n = 5 iimvavanyo ezizimeleyo. Uthelekiso lwezibalo lwenziwe kusetyenziswa uvavanyo lwe-ANOVA oluhamba ngendlela enye kunye neBonferroni post hoc. *p < 0.05; **p < 0.01; kunye ***p < 0.001
Ukuze siqonde ngakumbi impembelelo ye-IPA kwiprofayili ye-bioenergetic yeeseli ze-LX-2 ezisebenza nge-TGF-β1, sihlalutye i-phosphorylation ye-mitochondrial oxidative yi-OCR (Umzobo 3C, D). Iziphumo zibonise ukuba unyango lwe-TGF-β1 lunokunciphisa ukuphefumla okuphezulu, amandla okugcina umoya (i-SRC) kunye ne-BHI xa kuthelekiswa neqela lolawulo (Umzobo 3C, D). Ukongeza, unyango oludibeneyo lunciphise ukuphefumla kwe-basal, ukuvuza kweproton kunye nemveliso ye-ATP, kodwa i-SRC kunye ne-BHI zaziphezulu kakhulu kunezo ziphathwe nge-TGF-β1 (Umzobo 3C, D).
Sikwaqhube "uVavanyo lwePhenotype yamandla eSeli" olubonelelwe yisoftware yeSeahorse (Umzobo oNcedisayo 4A–D). Njengoko kubonisiwe kwiSupplementary Fig. 3B, zombini i-OCR kunye ne-ECAR metabolic potentials zinciphile emva konyango lwe-TGF-β1, nangona kunjalo, akukho mahluko ubonwe kumaqela onyango oludibeneyo kunye ne-IPA xa kuthelekiswa neqela lolawulo. Ngaphezu koko, amanqanaba e-basal kunye noxinzelelo lwe-OCR anciphile emva konyango oludibeneyo kunye ne-IPA xa kuthelekiswa neqela lolawulo (Umzobo oNcedisayo 4C). Okunomdla kukuba, iphethini efanayo yabonwa ngonyango oludibeneyo, apho kungekho tshintsho kumanqanaba e-basal kunye noxinzelelo lwe-ECAR olubonwe xa kuthelekiswa nonyango lwe-TGF-β1 (Umzobo oNcedisayo 4C). Kwii-HSC, ukuncipha kwe-phosphorylation ye-mitochondrial oxidative kunye nokukwazi konyango oludibeneyo ukubuyisela i-SCR kunye ne-BHI emva kokuvezwa kunyango lwe-TGF-β1 akuzange kutshintshe amandla e-metabolic (OCR kunye ne-ECAR). Xa zizonke, ezi ziphumo zibonisa ukuba i-IPA inokunciphisa i-bioenergetics kwi-HSCs, nto leyo ebonisa ukuba i-IPA inokubangela iprofayili yamandla aphantsi etshintsha i-phenotype ye-HSC iye ekungasebenzini (Umfanekiso Ongezelelweyo 4D).
Impembelelo ye-IPA kwi-mitochondrial dynamics iphandwe kusetyenziswa ukulinganiswa kwemilinganiselo emithathu ye-mitochondrial morphology kunye noqhagamshelo lwenethiwekhi kunye nokufakwa kwe-MTR staining (Umfanekiso 4 kunye noMfanekiso oNgezelelayo 5). Umfanekiso 4 ubonisa ukuba, xa kuthelekiswa neqela lolawulo, unyango lwe-TGF-β1 lunciphise indawo ephakathi yomphezulu, inani lesebe, ubude besebe lilonke, kunye nenani le-branch junction (Umfanekiso 4A kunye no-B) kwaye lutshintshe umlinganiselo we-mitochondria ukusuka kwi-spherical ukuya kwi-intermediate morphology (Umfanekiso 4C). Unyango lwe-IPA kuphela olunciphise ubuninzi be-mitochondrial kwaye lutshintshe umlinganiselo we-mitochondria ukusuka kwi-spherical ukuya kwi-intermediate morphology xa kuthelekiswa neqela lolawulo (Umfanekiso 4A). Ngokwahlukileyo koko, i-sphericity, ubude besebe eliphakathi, kunye nomsebenzi we-mitochondrial ovavanywe yi-mitochondrial membrane potential-dependent MTR (Umfanekiso 4A kunye no-E) azange zitshintshe kwaye ezi parameters azizange zahluke phakathi kwamaqela. Xa zizonke, ezi ziphumo zibonisa ukuba unyango lwe-TGF-β1 kunye ne-IPA lubonakala luguqula imo kunye nobukhulu be-mitochondrial kunye nobunzima benethiwekhi kwiiseli eziphilayo ze-LX-2.
I-IPA itshintsha amandla e-mitochondrial kunye nobuninzi be-DNA ye-mitochondrial kwiiseli ze-LX-2. A. Imifanekiso emele i-confocal yeeseli ze-LX-2 eziphilayo ezifakwe i-TGF-β1 (5 ng/ml) kunye ne-1 mM IPA iiyure ezingama-24 kwindawo engenazo i-serum ebonisa iinethiwekhi ze-mitochondrial ezidaywe yi-Mitotracker™ Red CMXRos kunye neenuclei ezidaywe luhlaza okwesibhakabhaka nge-DAPI. Yonke idatha iqulethe ubuncinci imifanekiso eli-15 kwiqela ngalinye. Sifumene imifanekiso eli-10 ye-Z-stack yohlobo ngalunye lwesampulu. Ulandelelwano ngalunye lwe-Z-axis lwaluqulathe izilayi ezingama-30, nganye inobukhulu obuyi-9.86 μm. Ibha yesikali: 10 μm. B. Izinto ezimeleyo (i-mitochondria kuphela) ezichongiweyo ngokusebenzisa umda oguquguqukayo kumfanekiso. Uhlalutyo lobungakanani kunye nothelekiso lonxibelelwano lwenethiwekhi ye-mitochondrial morphological lwenziwe kuzo zonke iiseli kwiqela ngalinye. C. Ukuphindaphinda kwemilinganiselo yemilo ye-mitochondrial. Amaxabiso akufutshane no-0 abonisa iimilo ezingqukuva, kwaye amaxabiso akufutshane no-1 abonisa iimilo ezingqukuva. D Umxholo weMitochondrial DNA (mtDNA) uchongiwe njengoko kuchaziwe kwiZixhobo kunye neeNdlela. Uhlalutyo lwe-E Mitotracker™ Red CMXRos lwenziwe nge-flow cytometry (iziganeko ezingama-30,000) njengoko kuchaziwe kwiZixhobo kunye neeNdlela. Idatha iboniswa njenge-mean ± SD, n = 3 iimvavanyo ezizimeleyo. Uthelekiso lwezibalo lwenziwe kusetyenziswa uvavanyo lwe-ANOVA oluhamba ngendlela enye kunye neBonferroni post hoc. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
Emva koko sihlalutye umxholo we-mtDNA kwiiseli ze-LX-2 njengophawu lwenani le-mitochondrial. Xa kuthelekiswa neqela lolawulo, umxholo we-mtDNA wonyuswe kwiqela eliphathwe nge-TGF-β1 (Umfanekiso 4D). Xa kuthelekiswa neqela eliphathwe nge-TGF-β1, umxholo we-mtDNA wehlisiwe kwiqela lonyango oludibeneyo (Umfanekiso 4D), nto leyo ebonisa ukuba i-IPA inokunciphisa umxholo we-mtDNA kwaye mhlawumbi inani le-mitochondrial kunye nokuphefumla kwe-mitochondrial (Umfanekiso 3C). Ngaphezu koko, i-IPA ibonakala ngathi iyawunciphisa umxholo we-mtDNA kunyango oludibeneyo kodwa ayizange ichaphazele umsebenzi we-mitochondrial olawulwa yi-MTR (Umfanekiso 4A–C).
Siphande unxulumano lwe-IPA namanqanaba e-mRNA yezakhi zofuzo ezinxulumene ne-fibrosis, i-apoptosis, ukusinda, kunye ne-mitochondrial dynamics kwiiseli ze-LX-2 (Umfanekiso 5A–D). Xa kuthelekiswa neqela lolawulo, iqela eliphathwe yi-TGF-β1 libonise ukwanda kokubonakaliswa kwezakhi zofuzo ezifana ne-collagen type I α2 chain (COL1A2), i-α-smooth muscle actin (αSMA), i-matrix metalloproteinase 2 (MMP2), i-tissue inhibitor ye-metalloproteinase 1 (TIMP1), kunye ne-dynamin 1-like gene (DRP1), ebonisa ukwanda kwe-fibrosis kunye nokusebenza. Ngaphezu koko, xa kuthelekiswa neqela lolawulo, unyango lwe-TGF-β1 lunciphise amanqanaba e-mRNA ye-nuclear pregnane X receptor (PXR), i-caspase 8 (CASP8), i-MAPKAPK3, i-inhibitor ye-B-cell α, i-enhancer ye-nuclear factor κ gene light peptide (NFκB1A), kunye ne-inhibitor ye-nuclear factor κB kinase subunit β (IKBKB) (Umfanekiso 5A–D). Xa kuthelekiswa nonyango lwe-TGF-β1, unyango oludibeneyo olune-TGF-β1 kunye ne-IPA lunciphise ukubonakaliswa kwe-COL1A2 kunye ne-MMP2, kodwa lonyusa amanqanaba e-mRNA e-PXR, TIMP1, B-cell lymphoma-2 (BCL-2), CASP8, NFκB1A, NFκB1-β, kunye ne-IKBKB. Unyango lwe-IPA lunciphise kakhulu ukubonakaliswa kwe-MMP2, i-Bcl-2-associated protein X (BAX), AKT1, i-optic atrophy protein 1 (OPA1), kunye ne-mitochondrial fusion protein 2 (MFN2), ngelixa ukubonakaliswa kwe-CASP8, NFκB1A, NFκB1B, kunye ne-IKBKB kwandisiwe xa kuthelekiswa neqela lolawulo. Nangona kunjalo, akukho mahluko ufunyenweyo ekubonakalisweni kwe-caspase-3 (CASP3), i-apoptotic peptidase activating factor 1 (APAF1), i-mitochondrial fusion protein 1 (MFN1), kunye ne-fission protein 1 (FIS1). Ngokudibeneyo, ezi ziphumo zibonisa ukuba unyango lwe-IPA luguqula ukubonakaliswa kwezakhi zofuzo ezinxulumene ne-fibrosis, i-apoptosis, ukusinda, kunye ne-mitochondrial dynamics. Idatha yethu ibonisa ukuba unyango lwe-IPA lunciphisa i-fibrosis kwiiseli ze-LX-2; kwangaxeshanye, luvuselela ukusinda ngokutshintsha i-phenotype iye kwi-inactivation.
I-IPA ilawula ukubonakaliswa kwe-fibroblast, i-apoptotic, i-viability, kunye ne-mitochondrial dynamics genes kwiiseli ze-LX-2. Ii-histogram zibonisa ukubonakaliswa kwe-mRNA ngokumalunga nolawulo lwe-endogenous (RPLP0 okanye i-PPIA) emva kokuba iiseli ze-LX-2 zifakwe i-TGF-β1 kunye ne-IPA kwindawo engena-serum kangangeeyure ezingama-24. I-A ibonisa ii-fibroblasts, i-B ibonisa iiseli ze-apoptotic, i-C ibonisa iiseli ezisindileyo, kwaye i-D ibonisa ukubonakaliswa kwe-mitochondrial dynamics gene. Idatha iboniswa njenge-mean ± standard deviation (SD), n = 3 iimvavanyo ezizimeleyo. Uthelekiso lwezibalo lwenziwe kusetyenziswa uvavanyo lwe-ANOVA oluhamba ngendlela enye kunye neBonferroni post hoc. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
Emva koko, utshintsho kubungakanani beseli (FSC-H) kunye nobunzima be-cytoplasmic (SSC-H) luhlolwe nge-flow cytometry (Umfanekiso 6A,B), kwaye utshintsho kwi-cell morphology emva konyango lwe-IPA luhlolwe nge-transmission electron microscopy (TEM) kunye ne-phase contrast microscopy (Umfanekiso oNcedisayo 6A-B). Njengoko bekulindelekile, iiseli kwiqela eliphathwe nge-TGF-β1 zanda ngobukhulu xa kuthelekiswa neqela lolawulo (Umfanekiso 6A,B), ebonisa ukwanda kweklasikhi kwe-rough endoplasmic reticulum (ER*) kunye ne-phagolysosomes (P), ebonisa ukusebenza kwe-hematopoietic stem cell (HSC) (Umfanekiso oNcedisayo 6A). Nangona kunjalo, xa kuthelekiswa neqela eliphathwe nge-TGF-β1, ubungakanani beseli, ubunzima be-cytoplasmic (Umfanekiso 6A,B), kunye nomxholo we-ER* wehlisiwe kwiqela lonyango oludibeneyo lwe-TGF-β1 kunye ne-IPA (Umfanekiso oNcedisayo 6A). Ngaphezu koko, unyango lwe-IPA lunciphise ubungakanani beseli, ubunzima be-cytoplasmic (IiFig. 6A,B), umxholo we-P kunye ne-ER* (Umzobo ongezelelweyo 6A) xa kuthelekiswa neqela lolawulo. Ukongeza, umxholo weeseli ze-apoptotic unyuke emva kweeyure ezingama-24 zonyango lwe-IPA xa kuthelekiswa neqela lolawulo (iintolo ezimhlophe, Umfanekiso ongezelelweyo 6B). Ngokudibeneyo, ezi ziphumo zibonisa ukuba i-1 mM IPA inokukhuthaza i-HSC apoptosis kwaye ibuyisele utshintsho kwiiparameter ze-morphological zeseli ezibangelwa yi-TGF-β1, ngaloo ndlela ilawula ubungakanani beseli kunye nobunzima, obunokunxulunyaniswa nokungasebenzi kwe-HSC.
I-IPA itshintsha ubungakanani beseli kunye nobunzima be-cytoplasmic kwiiseli ze-LX-2. Imifanekiso emeleyo yohlalutyo lwe-flow cytometry. Olu hlalutyo lusebenzise icebo lokugaya elithile kwiiseli ze-LX-2: i-SSC-A/FSC-A ukuchaza inani leeseli, i-FSC-H/FSC-A ukuchonga ii-doublets, kunye ne-SSC-H/FSC-H yobungakanani beseli kunye nohlalutyo lobunzima. Iiseli zafakwa kwi-TGF-β1 (5 ng/ml) kunye ne-1 mM IPA kwindawo engena-serum kangangeeyure ezingama-24. Iiseli ze-LX-2 zasasazwa kwi-quadrant esezantsi ekhohlo (SSC-H-/FSC-H-), i-quadrant ephezulu ekhohlo (SSC-H+/FSC-H-), i-quadrant esezantsi ekunene (SSC-H-/FSC-H+), kunye ne-quadrant ephezulu ekunene (SSC-H+/FSC-H+) yobungakanani beseli kunye nohlalutyo lobunzima be-cytoplasmic. B. Imo yeseli ihlalutywe nge-flow cytometry kusetyenziswa i-FSC-H (i-forward scatter, ubungakanani beseli) kunye ne-SSC-H (i-side scatter, i-cytoplasmic complexity) (iziganeko ezingama-30,000). Idatha iboniswa njenge-mean ± SD, n = 3 iimvavanyo ezizimeleyo. Uthelekiso lwezibalo lwenziwe kusetyenziswa uvavanyo lwe-ANOVA oluhamba ngendlela enye kunye ne-Bonferroni post hoc. *p < 0.05; **p < 0.01; ***p < 0.001 kunye ****p < 0.0001
Iimetabolites zamathumbu ezifana ne-IPA ziye zaba ngumxholo oshushu wophando, nto leyo ebonisa ukuba iithagethi ezintsha zinokufunyanwa kwi-gut microbiota. Kungoko ke kunomdla ukuba i-IPA, i-metabolite esiyidibanise ne-liver fibrosis ebantwini [15], ibonakaliswe njenge-compound enokubakho yokulwa ne-fibrotic kwiimodeli zezilwanyana [13, 14]. Apha, sibonisa okokuqala unxulumano phakathi kwe-IPA ye-serum kunye ne-global liver transcriptomics kunye ne-DNA methylation kubantu abakhulupheleyo abangenaso isifo seswekile sohlobo lwesibini (T2D), sigxininisa i-apoptosis, i-mitophagy kunye nokuphila ixesha elide, kunye ne-gene enokubakho ye-AKT1 elawula i-liver homeostasis. Enye into entsha yophando lwethu kukuba sibonise ukusebenzisana konyango lwe-IPA kunye ne-apoptosis, i-cell morphology, i-mitochondrial bioenergetics kunye ne-dynamics kwiiseli ze-LX-2, nto leyo ebonisa amandla aphantsi atshintsha i-phenotype ye-HSC iye kwi-inactivation, nto leyo eyenza i-IPA ibe ngumngeni onokubakho wokuphucula i-liver fibrosis.
Sifumanise ukuba i-apoptosis, i-mitophagy kunye nokuphila ixesha elide zezona ndlela zibalulekileyo ze-canonical ezityebiswe kwi-liver genes ezinxulumene ne-circulating serum IPA. Ukuphazamiseka kwenkqubo yokulawula umgangatho we-mitochondrial (MQC) kunokukhokelela ekungasebenzi kakuhle kwe-mitochondrial, i-mitophagy kunye ne-apoptosis, ngaloo ndlela kukhuthaza ukwenzeka kwe-MASLD[33, 34]. Ke ngoko, sinokuqikelela ukuba i-IPA isenokuba ibandakanyeka ekugcineni i-dynamics yeseli kunye nokuthembeka kwe-mitochondrial ngokusebenzisa i-apoptosis, i-mitophagy kunye nokuphila ixesha elide kwisibindi. Idatha yethu ibonise ukuba ii-genes ezimbini zaziqhelekile kuzo zonke iimvavanyo ezintathu: i-YKT6 kunye ne-AKT1. Kubalulekile ukuqaphela ukuba i-YKT6 yiprotheni ye-SNARE ebandakanyeka kwinkqubo yokuhlanganiswa kwe-membrane yeseli. Idlala indima kwi-autophagy kunye ne-mitophagy ngokwenza i-initiation complex ene-STX17 kunye ne-SNAP29 kwi-autophagosome, ngaloo ndlela ikhuthaza ukuhlanganiswa kwe-autophagosomes kunye ne-lysosomes[35]. Ngaphezu koko, ukulahleka komsebenzi we-YKT6 kubangela ukungasebenzi kakuhle kwe-mitophagy [36], ngelixa ukunyuka kwe-YKT6 kunxulunyaniswa nokuqhubela phambili kwe-hepatocellular carcinoma (HCC), okubonisa ukwanda kokusinda kweeseli [37]. Kwelinye icala, i-AKT1 yeyona gene ibalulekileyo esebenzisana nayo kwaye idlala indima ebalulekileyo kwizifo zesibindi, kubandakanya indlela yokubonisa ye-PI3K/AKT, umjikelo weseli, ukufuduka kweeseli, ukwanda, ukunamathela okugxileyo, umsebenzi we-mitochondrial, kunye nokukhutshwa kwe-collagen [38–40]. Indlela yokubonisa ye-PI3K/AKT esebenzayo inokusebenzisa iiseli ze-hematopoietic stem (HSCs), eziziiseli ezinoxanduva lokuvelisa i-extracellular matrix (ECM), kwaye ukungasebenzi kwayo kunokubangela ukwenzeka kunye nokuqhubela phambili kwe-liver fibrosis [40]. Ukongeza, i-AKT yenye yezinto ezibalulekileyo zokusinda kweeseli ezithintela i-p53-dependent cell apoptosis, kwaye ukusebenza kwe-AKT kunokunxulunyaniswa nokuthintela i-liver cell apoptosis [41, 42]. Iziphumo ezifunyenweyo zibonisa ukuba i-IPA isenokuba nefuthe kwi-apoptosis enxulumene ne-mitochondria yesibindi ngokuchaphazela isigqibo se-hepatocytes phakathi kokungena kwi-apoptosis okanye ukusinda. Ezi ziphumo zinokulawulwa yi-AKT kunye/okanye ii-YKT6 candidate genes, ezibalulekileyo kwi-homeostasis yesibindi.
Iziphumo zethu zibonise ukuba i-1 mM IPA ibangele i-apoptosis kwaye yehlisa ukuphefumla kwe-mitochondrial kwiiseli ze-LX-2 ngaphandle konyango lwe-TGF-β1. Kuyaphawuleka ukuba i-apoptosis yindlela ephambili yokusombulula i-fibrosis kunye nokusebenza kwe-hematopoietic stem cell (HSC), kwaye ikwangumcimbi obalulekileyo kwimpendulo yomzimba eguqukayo ye-fibrosis yesibindi [4, 43]. Ngaphezu koko, ukubuyiselwa kwe-BHI kwiiseli ze-LX-2 emva konyango oludibeneyo kubonelele ngolwazi olutsha ngendima enokubakho ye-IPA ekulawuleni i-mitochondrial bioenergetics. Phantsi kweemeko zokuphumla kunye nokungasebenzi, iiseli ze-hematopoietic zihlala zisebenzisa i-mitochondrial oxidative phosphorylation ukuvelisa i-ATP kwaye zinomsebenzi ophantsi we-metabolic. Kwelinye icala, ukusebenza kwe-HSC kuphucula ukuphefumla kwe-mitochondrial kunye ne-biosynthesis ukuhlawula iimfuno zamandla zokungena kwimeko ye-glycolytic [44]. Inyani yokuba i-IPA ayizange ichaphazele amandla e-metabolic kwaye i-ECAR ibonisa ukuba indlela ye-glycolytic ayibekwanga phambili kangako. Ngokufanayo, olunye uphando lubonise ukuba i-1 mM IPA ikwazile ukuguqula umsebenzi we-mitochondrial respiratory chain kwi-cardiomyocytes, umgca weseli ye-hepatocyte yomntu (i-Huh7) kunye neeseli ze-endothelial ze-umbilical vein yomntu (i-HUVEC); Nangona kunjalo, akukho mpembelelo ye-IPA efunyenweyo kwi-glycolysis kwi-cardiomyocytes, nto leyo ebonisa ukuba i-IPA inokuchaphazela i-bioenergetics yezinye iintlobo zeeseli [45]. Ke ngoko, sicinga ukuba i-1 mM IPA inokusebenza njenge-uncoupler yekhemikhali ethambileyo, kuba inokunciphisa kakhulu ukubonakaliswa kwe-fibrogen gene, i-cell morphology kunye ne-mitochondrial bioenergetics ngaphandle kokutshintsha ubungakanani be-mtDNA [46]. Ii-uncouplers ze-Mitochondrial zinokuthintela i-culture-induced fibrosis kunye nokusebenza kwe-HSC [47] kwaye zinciphise imveliso ye-mitochondrial ATP elawulwa okanye ebangelwa ziiproteni ezithile ezifana neeproteni ezidibanisayo (i-UCP) okanye i-adenine nucleotide translocase (i-ANT). Ngokuxhomekeke kuhlobo lweseli, le nto inokukhusela iiseli kwi-apoptosis kunye/okanye ikhuthaze i-apoptosis [46]. Nangona kunjalo, kufuneka uphando olongezelelweyo ukuze kucaciswe indima ye-IPA njenge-mitochondrial uncoupler ekuqalisweni kwe-hematopoietic stem cell inactivation.
Emva koko siphande ukuba utshintsho kwi-mitochondrial respiration lubonakala kwi-mitochondrial morphology kwiiseli ze-LX-2 eziphilayo. Okunomdla kukuba, unyango lwe-TGF-β1 lutshintsha i-mitochondrial proportional ukusuka kwi-spherical ukuya kwi-intermediate, kunye nokuncipha kwamasebe e-mitochondrial kunye nokwanda kokubonakaliswa kwe-DRP1, into ephambili ekuqhekekeni kwe-mitochondrial [48]. Ngaphezu koko, ukuqhekeka kwe-mitochondrial kunxulunyaniswa nobunzima benethiwekhi iyonke, kwaye utshintsho ukusuka ekuhlanganisweni ukuya ekuqhekekeni kubalulekile ekusebenzeni kwe-hematopoietic stem cell (HSC), ngelixa ukuthintela ukuqhekeka kwe-mitochondrial kukhokelela kwi-HSC apoptosis [49]. Ngoko ke, iziphumo zethu zibonisa ukuba unyango lwe-TGF-β1 lunokubangela ukwehla kobunzima benethiwekhi ye-mitochondrial ngokuncipha kwamasebe, okuxhaphake kakhulu kwi-mitochondrial fission enxulunyaniswa neeseli ze-hematopoietic stem cells (HSCs). Ngaphezu koko, idatha yethu ibonise ukuba i-IPA inokutshintsha i-mitochondria ukusuka kwi-spherical ukuya kwi-intermediate shape, ngaloo ndlela kunciphisa ukubonakaliswa kwe-OPA1 kunye ne-MFN2. Izifundo zibonise ukuba ukunciphisa i-OPA1 kunokubangela ukwehla kwamandla e-mitochondrial membrane kunye nokuqala i-apoptosis yeseli [50]. I-MFN2 yaziwa ngokuba yi-mediation mitochondrial fusion kunye ne-apoptosis [51]. Iziphumo ezifunyenweyo zibonisa ukuba ukungeniswa kweeseli ze-LX-2 yi-TGF-β1 kunye/okanye i-IPA kubonakala ngathi kuguqula imo kunye nobukhulu be-mitochondrial, kunye nemeko yokusebenza kunye nobunzima benethiwekhi.
Iziphumo zethu zibonisa ukuba unyango oludibeneyo lwe-TGFβ-1 kunye ne-IPA lunokunciphisa i-mtDNA kunye neeparameter ze-cell morphological ngokulawula ukubonakaliswa kwe-mRNA kwe-fibrosis, i-apoptosis kunye nee-genes ezinxulumene nokusinda kwiiseli eziphepha i-apoptosis. Enyanisweni, i-IPA yehlise inqanaba lokubonakaliswa kwe-mRNA ye-AKT1 kunye nee-genes ezibalulekileyo ze-fibrosis ezifana ne-COL1A2 kunye ne-MMP2, kodwa yonyusa inqanaba lokubonakaliswa kwe-CASP8, enxulunyaniswa ne-apoptosis. Iziphumo zethu zibonise ukuba emva konyango lwe-IPA, ukubonakaliswa kwe-BAX kwehla kwaye ukubonakaliswa kwe-mRNA kwee-subunits zosapho lwe-TIMP1, i-BCL-2 kunye ne-NF-κB kwanda, nto leyo ebonisa ukuba i-IPA inokukhuthaza imiqondiso yokusinda kwiiseli ze-hematopoietic stem (HSCs) eziphepha i-apoptosis. Ezi molekyuli zinokusebenza njengezibonakaliso zokusinda kwiiseli ze-hematopoietic stem ezisebenzayo, ezinokunxulunyaniswa nokubonakaliswa okwandisiweyo kweeproteni ezichasene ne-apoptotic (ezifana ne-Bcl-2), ukubonakaliswa okunciphileyo kwe-pro-apoptotic BAX, kunye nonxibelelwano oluntsonkothileyo phakathi kwe-TIMP kunye ne-NF-κB [5, 7]. I-IPA ivelisa iziphumo zayo nge-PXR, kwaye sifumanise ukuba unyango oludibeneyo nge-TGF-β1 kunye ne-IPA lonyusa amanqanaba okubonakaliswa kwe-PXR mRNA, nto leyo ebonisa ukucinywa kokusebenza kwe-HSC. I-activated PXR signaling iyaziwa ukuba ithintela ukusebenza kwe-HSC zombini kwi-vivo nakwi-vitro [52, 53]. Iziphumo zethu zibonisa ukuba i-IPA inokuthatha inxaxheba ekususweni kwe-activated HSCs ngokukhuthaza i-apoptosis, ukunciphisa i-fibrosis kunye ne-mitochondrial metabolism, kunye nokuphucula iimpawu zokusinda, eziziinkqubo eziqhelekileyo eziguqula i-activated HSC phenotype ibe yi-inactivated. Enye inkcazo enokwenzeka yendlela enokwenzeka kunye nendima ye-IPA kwi-apoptosis kukuba isusa i-mitochondria engasebenzi kakuhle ngokuyintloko nge-mitophagy (indlela yangaphakathi) kunye nendlela yokubonisa ye-TNF yangaphandle (Itheyibhile 1), edibene ngokuthe ngqo nendlela yokubonisa yokusinda ye-NF-κB (Umfanekiso ongezelelweyo 7). Okunomdla kukuba, ii-genes ezityebileyo ezinxulumene ne-IPA ziyakwazi ukukhuthaza imiqondiso ye-pro-apoptotic kunye ne-pro-survival kwindlela ye-apoptotic [54], nto leyo ebonisa ukuba i-IPA inokubangela indlela ye-apoptotic okanye ukusinda ngokusebenzisana nezi genes. Nangona kunjalo, indlela i-IPA ebangela ngayo i-apoptosis okanye ukusinda ngexesha lokusebenza kwe-HSC kunye neendlela zayo zokusebenza azikacaci.
I-IPA yi-metabolite ye-microbial eyenziwe kwi-tryptophan yokutya nge-gut microbiota. Izifundo zibonise ukuba ineempawu zokulawula ukudumba, i-antioxidant, kunye ne-epigenetic kwindawo yamathumbu.[55] Izifundo zibonise ukuba i-IPA inokuguqula ukusebenza komqobo wamathumbu kwaye inciphise uxinzelelo lwe-oxidative, olunokuba negalelo kwimiphumo yayo ye-physiological yendawo.[56] Enyanisweni, i-IPA ithuthwa iye kwizitho ezijoliswe kuzo ngokujikeleza kwegazi, kwaye ekubeni i-IPA inesakhiwo esifanayo se-metabolite kunye ne-tryptophan, i-serotonin, kunye ne-indole derivatives, i-IPA inezenzo ze-metabolic ezikhokelela kwi-metabolic fates ekhuphisanayo.[52] I-IPA inokukhuphisana ne-tryptophan-derived metabolites kwiindawo zokubopha kwi-enzymes okanye kwi-receptors, okunokuphazamisa iindlela eziqhelekileyo ze-metabolic. Oku kugxininisa imfuneko yophando olongezelelweyo kwi-pharmacokinetics yayo kunye ne-pharmacodynamics ukuze kuqondwe ngcono i-window yayo yonyango.[57] Kusafuneka kubonwe ukuba oku kunokwenzeka na nakwii-hematopoietic stem cells (HSCs).
Siyavuma ukuba uphando lwethu lunemida ethile. Ukuze sihlolisise ngokukodwa unxulumano olunxulumene ne-IPA, sizikhuphe ngaphandle izigulana ezine-type 2 diabetes mellitus (T2DM). Siyavuma ukuba oku kunciphisa ukusetyenziswa ngokubanzi kweziphumo zethu kwizigulana ezine-type 2 diabetes mellitus kunye nesifo sesibindi esiphambili. Nangona uxinano lwe-IPA kwi-serum yomntu luyi-1–10 μM [11, 20], uxinano lwe-1 mM IPA lukhethwe ngokusekelwe kuxinano oluphezulu olungenatyhefu [15] kunye nesantya esiphezulu se-apoptosis, kungekho mahluko kwipesenti yabantu beeseli ezifileyo. Nangona amanqanaba e-IPA angaphezulu kwe-physiological asetyenziswe kolu phononongo, okwangoku akukho mvumelwano malunga nedosi esebenzayo ye-IPA [52]. Nangona iziphumo zethu zibalulekile, ikamva le-metabolic ebanzi ye-IPA lihlala liyindawo esebenzayo yophando. Ngaphezu koko, iziphumo zethu malunga nonxulumano phakathi kwamanqanaba e-IPA e-serum kunye ne-DNA methylation ye-liver transcripts azifunyanwanga kuphela kwiiseli ze-hematopoietic stem (HSCs) kodwa nakwiithishu zesibindi. Sikhethe ukusebenzisa iiseli ze-LX-2 zabantu ngokusekelwe kwiziphumo zethu zangaphambili ezivela kuhlalutyo lwe-transcriptome zokuba i-IPA inxulunyaniswa nokusebenza kwe-hematopoietic stem cell (HSC) [15], kwaye ii-HSC zezona seli ziphambili ezibandakanyekayo ekuqhubekeni kwe-fibrosis yesibindi. Isibindi senziwe ngeentlobo ezininzi zeeseli, ngoko ke ezinye iimodeli zeeseli ezifana nenkqubo ye-hepatocyte-HSC-immune cell co-culture edityaniswe nokusebenza kwe-caspase kunye nokuqhekeka kwe-DNA kunye nendlela yokusebenza kubandakanya nenqanaba leproteni kufuneka ziqwalaselwe ukuze kufundwe indima ye-IPA kunye nokusebenzisana kwayo nezinye iintlobo zeeseli zesibindi.